Next Generation Sequencing FAQs

RNA-SEQ

RNA-Seq is a method for transcriptome profiling that uses next generation sequencing technologies. RNA-Seq provides a comprehensive, quantitative, and unbiased view of RNA sequences within every sample.

Microarray technology utilizes a pre-defined set of probes to capture and quantify specific RNA sequences. This means that microarrays are capable of detecting only a pre-selected set of transcripts. RNA-Seq relies on next generation sequencing technologies, enabling the identification and quantification of any RNA sequence in the sample.

The number of reads required depends upon the genome size, the number of known genes, and transcripts. GENEWIZ recommends 10 million reads for small genomes (i.e. bacteria) and 30 million reads for large genomes (i.e. human, mouse). A larger number of reads will generate more sequencing data, which increases the chances of detecting genes that are expressed at lower levels. Please note: A sufficient amount of sequencing data is necessary to ensure the quality of downstream data analysis results; an insufficient number of reads may lead to generating results that cannot be analyzed.

GENEWIZ Project Management, a team of dedicated technical experts, is available to assist with experimental design and to answer any questions for RNA-Seq projects. Project Management is accessible via email or by phone at +1-908-222-0711 ext 1 from 8:00 a.m. – 6:00 p.m. (ET).

International orders: Please inform shipping agents that your package is perishable. Please send GENEWIZ the tracking information for your package upon shipment. Because international shipments process through Customs, please retain a sufficient amount of your samples in the event you need to resubmit materials to GENEWIZ.

Yes, GENEWIZ accepts ds-cDNA as a starting material for RNA-Seq projects. Please note, GENEWIZ is unable to provide full quality control when using ds-cDNA. We request at least 1 µg of ds-cDNA with a concentration of ≥50 ng/µl.

Yes, GENEWIZ accepts mRNA as a starting material. However, we will be unable to do a quality check before beginning the library construction process. We request > 20 ng of mRNA to start. Samples should be eluted or re-suspended in nuclease-free water.

GENEWIZ generally uses a poly-A selection method to enrich RNA transcripts for eukaryotic samples. In some cases, it is more effective to deplete ribosomal RNA (rRNA) as an alternative method to poly-A selection. For example, total RNA from species with a large amount of RNA would benefit from using rRNA depletion. Furthermore, if you need to detect several types of non-coding elements, such as long and short ncRNAs, we recommend using the rRNA depletion method.

GENEWIZ guarantees delivery of the number of reads selected for your samples. As a guideline, the delivery guarantee is 120 million reads per lane on the Illumina HiSeq 2500 in rapid run mode and 250 million reads per lane in high output mode.

Client confidentiality and protection of Intellectual Property (IP) are of the utmost importance at GENEWIZ. Clients take confidence in the security and privacy of all projects completed with GENEWIZ. For more information, please reference the GENEWIZ Confidentiality Policy.

WHOLE GENOME SEQUENCING

De novo assembly can present a bioinformatics challenge, in particular to close any gaps remaining following sequence assembly.

During the assembly process, you end up with large DNA sequences known as contigs formed from overlapping sequence reads. Since next generation sequencing (NGS) works by sequencing fragmented nucleic acid, gaps will exist between contigs.

The size of full genomes can be very large. Targeted resequencing lets you remove areas that you are not interested in knowing more about so that your initial target size is smaller. Since the output remains constant for a particular NGS platform and configuration, a smaller target size gives you the flexibility of multiplexing more samples into the same run or sequencing at a higher depth of coverage.

Increased ability to multiplex makes the project more cost-effective since you are sequencing more samples in a single run (significantly decreasing the per sample cost).

Higher depth of coverage gives the assay more sensitivity. This higher sensitivity allows you to detect low-frequency, rare mutations more effectively. Also, it allows for resolving more complex (e.g., GC- or AT-rich) genomic regions.

Exome sequencing is a form of targeted resequencing. However, the target size is significantly larger than most custom targeted assays. For this reason, the same benefits apply as compared to whole genome sequencing.

There are primarily two types of targeted resequencing: targeted enrichment and amplicon sequencing. Targeted enrichment captures regions of interests with baits following creation of the full genomic library. By contrast, amplicon sequencing amplifies the target regions directly from the unfragmented genomic DNA in a highly multiplexed PCR reaction. Further, there are multiple targeted enrichment and amplicon sequencing technologies designed by different companies, including Illumina, Agilent, and Life Technologies.

The assay design process can range from relatively straightforward to extremely complex.

Some key elements of the design process include:

Deciding between amplicon sequencing and targeted enrichment

Deciding on the best amplicon or enrichment chemistry/technology

Determining the best design for the particular regions/genes (e.g., a GC-rich or complex set of regions would be handled differently than a simple one)

GENEWIZ possesses extensive experience with targeted assay design, which has been demonstrated with the development of multiple in-house, proprietary panels optimized for specific applications (e.g., OncoGxOne™ Discovery cancer panels, which are optimized for cancer research).

Whole genome sequencing requires an extremely high amount of sequencing throughput to generate even a moderate depth of coverage. The data generated, while comprehensive, will not allow the detection of mutations with as much sensitivity as a targeted approach.

Exome sequencing is a targeted approach that targets approximately 1-2% of the whole genome. As a result, you can generate more data, and therefore a higher depth of coverage to achieve more sensitivity.

Cancer panels target only a small percentage of what exome sequencing targets. The depth of coverage is therefore compounded even more, which makes the sensitivity of the assay high enough to effectively detect even the very rare mutations. This is crucial for cancer, because somatic mutations--which have been demonstrated to often be cancer drivers--tend to occur at a very low frequency.

Hot Spot Cancer Panels target regions of 48-50 genes that have been well-characterized as mutational hot spots. This is often a very small portion of the exons of those genes. The genes assayed are general to a number of different cancer types. The technology utilized for targeting is amplicon sequencing, which hybridizes pre-designed primers to flank the regions of interest directly to unfragmented genomic DNA in a highly multiplexed PCR reaction. Hot Spot Cancer Panels can provide information about point mutations and small insertions/deletions (indels).

OncoGxOne™ Discovery cancer panels target all exons, all UTRs, and introns for which there are known translocation breakpoints of ~150-400 genes. The precise number of genes varies between the 19 panels. Each panel is specific to one cancer type. The technology utilized for targeting is targeted enrichment, which hybridizes pre-designed biotinylated baits to a fully prepared genomic library in order to pull down the regions of interest. OncoGxOne™ Discovery cancer panels can provide information about point mutations, indels, copy number variations (CNVs), and gene fusions.

The two options have their benefits and drawbacks. For example, OncoGxOne™ Discovery panels are more comprehensive, but will take longer to generate data and will have a higher cost.

An expert Project Manager is happy to discuss these options with you in order to determine which method would be optimal for your project and situation.

A typical OncoGxOne Discovery cancer panel contains approximately 250 cancer-specific genes (including all known cancer driver genes), varying from panel to panel, ranging from ~150 to ~350 genes. The cancer gene selection for each panel is done via very extensive literature and database mining, followed by expert-curation and extensive review to ensure the scientific validity, comprehensiveness, and relevance. The data sources used for data mining include all major cancer gene-containing databases, such as COSMIC, CGC, TCGA, CGAP/Mitelman, OMIM, HMD, TTD, and DrugBank, etc., publicly available cancer panels, and major publications.

Specifically designed: Each OncoGxOne™ Discovery cancer panel is specifically designed to assay a distinct cancer, while the off-the-shelf cancer panels are general gene panels, containing many genes which may not be relevant to the cancer type of interest.

More comprehensive: A typical OncoGxOne™ Discovery cancer panel contains ~250 cancer genes, while the off-the-shelf cancer panels contain only ~50 genes. In addition, among these ~50 genes, many may not be relevant to the cancer type of interest.

Detects more types of genomic aberrations: Because of our proprietary panel design and bioinformatics techniques, OncoGxOne™ Discovery cancer panels can detect all four types of genomics alterations (point mutation, Indel, gene fusion, CNV), while the off-the-shelf cancer panels can only detect point mutations and Indels.

In order to detect gene fusions, GENEWIZ designs SureSelect probes near every potential known fusion site, and uses effective bioinformatics techniques to identify the gene fusion locations and partners. To assess the CNV (copy number variations) detection, we use control genes and "background controls" generated from the pooled customer sample data, together with proprietary bioinformatics methods. We can also use control samples (usually a blood sample, provided by customer) from the same subject to help improve the accuracy. In addition, we provide qPCR service as another CNV assay method for further verification.

Yes, we can provide the gene lists for all of our cancer panels to potential customers. We can also highlight those genes with their intron regions covered. The gene lists will be regularly updated, and we can include any additional genes upon customers' request.

Exome Sequencing

Exome sequencing is the targeted enrichment and subsequent sequencing of the whole exome. Exome sequencing is potentially the most powerful tool available to the research community for the identification of genetic variations associated with a phenotype, such as a disease.

Whole genome sequencing requires an extremely high amount of sequencing throughput to generate even a moderate depth of coverage. The data generated, while comprehensive, will not allow detection of mutations with as much sensitivity as a targeted approach. Paired with current sequencing technologies, exome sequencing is the most cost-effective and efficient solution.

A large portion of relevant mutations occur in the exome. In fact, the exome contains as many as 85% of disease-related mutations. Covering less than 2% of the whole genome, exome sequencing requires only 1/50th of the sequencing throughput to generate the same depth of coverage. The lower sequencing throughput required by exome sequencing provides flexible experimental options:

1. Maintain the same depth of coverage and multiplex more samples into the same lane, significantly decreasing total project cost;

2. Increase the depth of coverage to facilitate the detection of rare, low-frequency mutations;

GENEWIZ has proven our steadfast commitment to superior service throughout the years, becoming established as a global leader in DNA sequencing and genomics. Further, we have extensive experience and expertise in next generation sequencing techniques, including exome sequencing. An expert Project Manager can assist you in determining specifications for optimizing your project.

Our targeted 16S-EZ assay can typically detect down to genus level. Sensitivity is ultimately dependent on the classification of the organism in the data analysis database and the abundance of the taxa in the sample itself.

In addition to industry leading turnaround time, 16S-EZ uses a proprietary primer pool to target the hypervariable V3 and V4 regions of the 16S rRNA gene, which has been shown to be more sensitive and accurate compared to V4-only sequencing.